EdU labeling assay

EdU Staining for Cells

1. Pulse cells by diluting stock 10mM EdU (1,000X, so 1 uL EdU per 1 mL media) in media at 37°C (incubator) for an appropriate amount of time.
2. Wash cells 1x with PBS (each wash is 3 min)
3. Fix with 4% PFA for 10 min
4. Wash cells 2x with PBS
5. Permeabilize cells with 0.3% TritonX-100 in PBS for 10 min
6. Wash cells 2x with PBS
7. During permeabilization, freshly prepare 200 mg/mL of Ascorbic Acid by dissolving in PBS (to 1 mL of PBS at 0.2 g of Ascorbic Acid)
8. Freshly prepare EdU Staining Mix: for a 6-well plate add 500 uL to each well, so prepare master mix accordingly

    9. Incubate in dark at room temperature for 30 min (hereon, always cover cells with aluminum foil)

  10. Wash cells 2x with PBS

  11. Here, proceed with blocking with 5% GS for 1 hr and addition of primary antibody for O/N incubation followed by washes and secondary antibody incubation. OR

  12. Stain with DAPI (dilute 5 mg/mL DAPI 10,000X in PBS) for 5 min at room temperature

  13. Wash cells 2x with PBS 

  14. Image or store at 4°C covered with aluminum foil